Fish Physiology and Biochemistry vol. 11 no. 1-6 pp 71-76 (1993) Kugler Publications, Amsterdam/New York
The regulatory effects of thyrotropin-releasing hormone on growth hormone secretion from the pituitary of common carp in vitro Xin-Wei Lin l , Hao-Ren Lin 1 and Richard E. Peter 2 'Department of Biology, Zhongshan University, Guangzhou, P.R. China. 510275; 2 Department of Zoology, University of Alberta, Edmonton, Alberta, Canada. T6G 2E9
Keywords: TRH, growth hormone, somatostatin, apomorphine, extracellular calcium, pituitary fragment, common carp
Abstract The effects of thyrotropin-releasing hormone (TRH) on growth hormone (GH) and gonadotropin (GtH) release, and the influences of somatostatin (SRIF), the dopamine agonist apomorphine (APO) and extracellular calcium on basal and TRH-induced GH release were examined using an in vitro perifusion system for pituitary fragments of common carp (Cyprinus carpio). Five minute pulses of different dosages of TRH stimulated a rapid and dose-dependent increase in GH release from the perifused pituitary fragments with an ED 50 of 9.7 + 2.3 nM. TRH was ineffective on GtH release. SRIF significantly inhibited basal and TRHinduced GH release from the perifused pituitary fragments, and the effects of SRIF were dose-dependent. APO induced a dose-dependent increase in basal and TRH-stimulated GH release from the perifused pituitary fragments. Increasing the concentrations of extracellular calcium from 0 mM to 1.25 mM resulted in an increase in basal and TRH-induced GH release. The high dose of calcium (6.25 mM) caused a slight decrease in basal and TRH-induced GH release compared with those at a concentration of 1.25 mM.
Resume Les effets de la thyrotropine (TRH) sur la scretion d'hormone de croissance (GH) et de gonadotropine (GTH), et de la somatostatine (SRIF), de l'apomorphine (APO), antagoniste dopaminergique, et du calcium extracellulaire sur les scr6tions basale et stimul6e de GH ont ete 6tudi6es in vitro par p6rifusion, de fragments d'hypophyses de carpe (Cyprinus carpio). Des applications de 5 minutes de TRH a diff6rentes concentrations induisent une stimulation rapide et dose d6pendante de la s6cr6tion de GH (ED 50 = 9.7 + 2.3 nM). Le TRH est sans effet sur la scr6tion de GTH. Le SRIF inhibe la scr6tion basale de GH ainsi que la rsponse hypophysaire a l'action du TRH. Son action est dose d6pendante. L'apomorphine induit une augmentation dose d6pendante de la s6cr6tion basale de GH et potentialise l'action du TRH sur la stimulation de la s6cr6tion de GH. Des effets equivalents sont induits par des concentrations croissantes de calcium extra cellulaire de 0 a 1.2 mM, alors qu'A une concentration de 6.25 mM des effets opposes sont obtenus. Introduction Thyrotropin-releasing hormone (TRH), a physiological stimulator of thyrotropin (TSH) and prolac-
tin secretion in mammals, has also been implicated as a stimulator of growth hormone (GH) release in certain pathological states and in perifusion with anterior pituitary cells of normal rat (for review, see
72 Harvey 1990). In birds, TRH is a major stimulatory factor of GH secretion (Hall et al. 1986; Harvey 1990), and TRH has also been reported to stimulate GH secretion in frogs (Hall and Chadwick 1984) and turtles (Denver and Licht 1989). A stimulatory effect of TRH on GH release has been suggested in the sailfin molly based on electrophoretic measurements of GH (Wigham and Batten 1984) and TRH stimulates GH release from perifused fragments of the goldfish pituitary (Trudeau et al. 1992). However, the interactions of somatostatin (SRIF), neurotransmitters or extracellular calcium on the actions of TRH on GH release in teleosts have not been investigated. In the present study, the effects of TRH on in vitro GH and gonadotropin (GtH) secretion in common carp were investigated. The influences of somatostatin (SRIF), the dopamine agonist apomorphine (APO) and extracellular calcium on basal and TRH-induced GH release were also examined. Materials and methods Common carp (Cyprinus carpio L.), 337-628 g of body weight, were purchased from local suppliers (Guangdong Province, P.R. China). Sexually recrudescing fish (male and female) were obtained in September (gonadosomatic index, GSI = 10.5 + 4.27o) for the dose-response experiments, and in November (GSI = 14.8 + 1.5%) for the other experiments. Fish were held in indoor recirculating 2501 aquaria at room temperature (18-28 0 C), with exposure to a natural photoperiod (Guangzhou, Guangdong Province, P.R. China). Experiments were conducted using a column perifusion system for pituitary fragments of common carp, similar to that used for goldfish pituitary fragments (Mackenzie et al. 1984; Habibi et al. 1989), with minor modifications. Briefly, the pituitaries were removed and fragments were prepared (< 1 mm3 ). Fragments equivalent to one half-pituitary were placed between two layers of Cytodex microcarrier beads (Sigma, St. Louis, MO, USA) in a 0.3 ml perifusion chamber. The fragments were perifused overnight (8-10h) with Medium 199 containing Hank's salts and Lglutamine (Gibco), 25 mM Hepes (Sigma) and 56
U/ml Nystatin (Sigma), at a flow rate of 5 ml/h. At 2h prior to the experiment, the medium was switched to Hank's balanced salt solution supplemented with 25 mM Hepes and 0.1% bovine serum albumin (Sigma) (HBSS, [Ca2 +] = 1.25 mM), or calcium-deficient HBSS in the fourth set of experiments at 19 + I°C, and the flow rate increased to 15 ml/h. Fractions were collected every 5 min., TRH (provided by the Laboratory of Reproductive Biology, Institute of Zoology, Academia Sinica) and SRIF (Sigma) were diluted with HBSS from stock solution (100 zM in HBSS), immediately before use. APO (Sigma) was dissolved in 0.8% NaCl with 0.1% sodium metabisulphite and diluted with HBSS just before use. In the dose-response study, the pituitary fragments were exposed to 5 min. pulses of increasing or decreasing concentrations of TRH (0.1, 1, 10, 100 and 1000 nM or 5, 50, 500, 5000 and 10000 nM) at 60 min. intervals. A total of 10 dosages of TRH were tested, each with six columns from three separate perifusion runs. The influences of APO on basal and TRHinduced GH release were studied in the second set of experiments. The pituitary fragments were exposed to three 5 min. pulses of increasing or decreasing concentrations of TRH (1, 10 and 100 nM) every 60 min. in combination with either HBSS alone, or 10, 100 or 1000 nM APO respectively. Each dose of TRH was tested with 4 column replicates from separate perifusion runs. The influences of SRIF on basal and TRHinduced GH release were studied in the third set of experiments. Three 5 min. pulses of 50 nM TRH were introduced at 60 min. intervals in the same perifusion column (four columns in each run, with two replicate runs). After the first pulse of TRH the pituitary fragments were perifused with 100 nM SRIF (in two columns) or in 10 nM SRIF (in another two columns), each lasting 60 min. and commencing 20 min. prior to the addition of the second TRH pulse. At the end of perifusion with SRIF, the fragments were perifused with HBSS alone for 25 min., and then the third pulse of TRH was introduced during continuous perifusion with HBSS. The influences of extracellular calcium on basal
73 and TRH-induced GH release were studied in the fourth set of experiments. The pituitary fragments were exposed to three 5 min. pulses of increasing or decreasing concentrations of TRH (1, 10 and 100 nM) every 60 min. in combination with Ca 2 +-deficient HBSS, or HBSS containing 0.25 mM, 1.25 mM and 6.25 mM Ca 2 + , respectively. Each dose of TRH was tested with 4 column replicates from separate perifusion runs. Calcium-deficient HBSS was HBSS without addition of Ca 2 + salts and containing 100 tM EGTA (Sigma). GtH and GH concentrations in the perifusates were determined by RIAs specific for common carp GtH-II (Peter et al. 1984; Van Der Kraak et al. 1992) and GH (Marchant et al. 1989), respectively. The hormone responses to each pulse of TRH were quantified, according to Habibi et al. (1989), and expressed as a percentage of the average basal hormone levels (prepulse) in the three fractions (15 min.) collected immediately preceding each pulse (% of prepulse). To calculate basal hormone levels, in the second set of experiments, the basal GH levels in the presence of different concentrations of APO were normalized as a percentage of the average GH levels over the 30 min. period before treatment with APO ( of pretreatment); in the third set of experiments, the basal GH levels in the presence of SRIF and HBSS alone preceding the second and third TRH pulses respectively were expressed as a percentage of the average GH level preceding the first pulse ( of 1st prepulse), and the first prepulses were quantified at 100%; in the fourth set of experiments, the average basal GH levels of prepulses in the presence of different concentrations of Ca 2 + were expressed as a percentage of the average GH levels over the 30 min. period during perifusion of calcium-deficient HBSS (o of pretreatment). The data were subjected to one-way analysis of variance followed by Duncan's multiple range test. The half-maximal effective dose (ED 50 ) value of TRH in stimulating GH release was calculated according to Karber (1931).
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TRH (nM) Fig. 1. Representative profile of GH release from perifused pituitary fragments of common carp in response to increasing doses of TRH administered as 5 min. pulses (black bar) every 60 min. (upper panel), and the dose-response curve of TRH in stimulation of GH and the effects of TRH on GtH secretion (lower panel). All values in the dose-response are mean + SEM of 6 observations from 3 separate perifusion runs. The ED 50 value of TRH on GH release is 9.7 + 2.3 nM. The average basal GtH and GH levels in these perifusion columns were 33.3 + 3.1 ng/ml and 126.2 + 34.7 ng/ml respectively.
Results Effects of TRH stimulating GH and GtH release Exposure of the sexually recrudescing common carp pituitary fragments to 5 min. pulses of increasing (upper panel, Fig. 1) or decreasing (not shown) doses of TRH resulted in a rapid and dosedependent stimulation of GH secretion. The ED 50 (mean approximate error) was estimated as 9.7 + 2.3 nM using 10 doses of TRH in order of increasing and decreasing concentration. TRH did not affect GtH release (lower panel, Fig. 1). Influences of APO on basal and TRH-induced GH secretion APO stimulated an increase in basal GH release (prepulse) in a dose-dependent manner (upper
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Fig. 2. Influences of APO on basal (upper panel) and TRHinduced (lower panel) GH release from perifused pituitary fragments of common carp. Each value is mean ± SEM of 4 observations from 4 individual perifusion runs. The average pretreatment GH level was 94.0 + 18.1 ng/ml. Similar basal GH levels (upper panel), or similar GH responses to the same dose of TRH in the presence of different doses of APO were identified by the same underscore (p < 0.05).
panel, Fig. 2). The high doses of APO (100, 1000 nM) significantly potentiated the actions of TRH (lower panel, Fig. 2); however, there were no significant differences between the GH release responses to pulses of TRH given alone and given in the presence of 10 nM APO. Influences of SRIF on basal and TRH-induced GH release Pulses of TRH (50 nM) alone stimulated a rapid GH release. The presence of 10 or 100 nM SRIF resulted in a significant reduction in basal and 50 nM TRH-induced GH release (upper panel, Fig. 3). SRIF (10 nM) partially blocked the TRH-induced GH release, and did not affect the GH release response to the third pulse of TRH (control) and the basal GH levels preceding this pulse (lower panel, Fig. 3); 100 nM SRIF completely blocked TRHinduced GH release (lower panel, Fig. 3) and significantly reduced the basal GH levels preceding the third TRH pulse (upper panel, Fig. 3).
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Fig. 3. Influences of SRIF on basal (upper panel) and TRHinduced (lower panel) GH release from perifused pituitary fragments of common carp. Each value represents mean + SEM of 4 observations from 2 separate perifusion runs. The average GH levels of the first prepulses was 108.6 + 26.1 ng/ml. The similar basal GH levels (upper panel), or GH release responses to TRH in the presence or absence of SRIF are identified by the same underscore (p < 0.05).
Influences of extracellular calcium on basal and TRH-induced GH secretion Basal GH levels were significantly increased with the elevation of concentrations of extracellular medium Ca 2 + from 0 mM to 1.25 mM; however, 6.25 mM Ca 2 + caused a slight decrease (insignificant) in basal levels compared to those at 1.25 mM 34.1 ng/ml) Ca 2 + . The basal GH levels (115.8 in the presence of 1.25 mM Ca 2 + (normal medium) were about four times the levels of basal GH (29.2 + 7.4 ng/ml) in the absence of Ca 2 + (Ca2+-deficient HBSS) (upper panel, Fig. 4). TRH-induced GH release responses were also significantly increased in the presence of increased concentrations of medium Ca 2 + (lower panel,
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TRH (nM) 2+ concentration on basal Fig. 4. Effects of extracellular Ca (upper panel) and TRH-induced (lower panel) GH release from perifused pituitary fragments of common carp. Each value is mean + SEM of 4 observations from 4 separate perifusion runs. 2 The average basal GH level in Ca +-deficient HBSS was 29.2 + 7.4 ng/ml. The similar basal GH levels (upper panel) or GH release responses to the same dose of TRH in the presence of different doses of Ca 2 + (lower panel) are identified by the same underscore (p < 0.05).
Fig. 4). In the absence of Ca 2 + , the GH-releasing actions of TRH were negligible. The high dose of Ca 2 + (6.25 mM) caused a reduction in the GH release responses to 10 nM, but not 1 and 100 nM TRH compared to the responses in the presence of 1.25 mM (lower panel, Fig. 4).
Discussion In the present study, we demonstrated that TRH can stimulate GH release via direct action on the pituitary of common carp. The potency of TRH in stimulating GH secretion from perifused pituitary fragments of common carp (sexually recrudescing, September) was similar to that in sexually regressed goldfish (Trudeau et al. 1992). TRH receptors in
brain and pituitary of goldfish have been partially characterized, with an apparent dissociation constant of 2-4 nM (Burt and Ajah 1984), which is in a similar range to the ED 50 estimate for TRHinduced GH secretion in the present study. The present results also demonstrate that TRH is ineffective on GtH release, indicating the GH response to TRH was specific. Present results further support the hypothesis that SRIF acts as the GH release-inhibiting factor in teleosts (Marchant et al. 1987). This is the first demonstration that the stimulatory actions of TRH on GH secretion in a teleost fish can be inhibited by SRIF, supporting the presence of a physiological role for TRH in GH secretion in teleosts. The influences of dopamine on GH-releasing actions of TRH in vertebrates are not clear. In the present study, the non-selective dopamine agonist APO induced a dose-dependent increase in basal GH release, similar to earlier findings in common carp (unpublished results) and goldfish (Chang et al. 1990). Dopamine acts via D-1 type receptors on somatotrophs to stimulate GH release in goldfish (Chang et al. 1990; Wong et al. 1992), and presumably is one of several GH-releasing factors. APO significantly increased TRH-stimulated GH release in the present study, suggesting the presence of dopaminergic regulation on GH secretion in common carp. In mammals and birds, several reports have suggested the involvement of a cAMP-independent, calcium-dependent mechanism underlying the stimulatory effects of TRH on GH secretion (Hart et al. 1988; Hall et al. 1985). Present results demonstrated that the mechanism of TRH action on in vitro GH release in common carp is Ca 2 + dependent, and the GH responses to TRH are somewhat reduced by a high concentration of Ca2 + (6.25 mM). Inhibition of neuropeptide-induced GH secretion by a supraphysiological dose of Ca 2 + was also reported for TRH in chicken (Hall et al. 1985) and for GHRH in mammals (Hart et al. 1988), but the mechanism for this inhibition remains to be determined. The present results also showed that basal GH release is dependent on extracellular Ca 2 +, consistent with the idea that extracellular Ca2 + plays an important role in mediating basal
76 GH secretion in the goldfish (Chang and De Leeuw 1990) and in other vertebrates (Hall et al. 1985; Stojilkovic et al. 1988).
Acknowledgements This work was supported by the Doctoral Fund from the National Education Committee of China to H.R. Lin and by grant 3-P-83-1101 from the International Development Research Center of Canada to H.R. Lin and R.E. Peter. We are grateful to J.P. Chang and H.R. Habibi for their help in the establishment of the pituitary perifusion system in H.R. Lin's laboratory. We also thank H. Kawauchi (Kitsato University, Japan) for the gift of purified common carp GH and GtH used in RIAs.
References cited Burt, P.R. and Ajah, M.A. 1984. TRH receptors in fish. Gen. Comp. Endocrinol. 53: 135-142. Chang, J.P. and De Leeuw, R. 1990. In vitro goldfish growth hormone responses to gonadotropin-releasing hormone: possible role of extracellular calcium and arachidonic acid metabolism? Gen. Comp. Endocrinol. 80: 155-164. Chang, J.P., Yu, K.L., Wong, A.O.L. and Peter, R.E. 1990. Differential actions of dopamine receptor subtypes on gonadotropin and growth hormone release in vitro in goldfish. Neuroendocrinology 51: 664-674. Denver, R.J. and Licht, P. 1989. Neuropeptides influences in vitro pituitary hormone secretion in hatchling turtles. J. Exp. Zool. 251: 306-315. Habibi, H.R., Marchant, T.A., Nahorniak, C.S., Van Der Loo, H., Peter, R.E., Rivier, J.E. and Vale, W.W. 1989. Functional relationship between receptor binding and biological activity for analogs of mammalian and salmon gonadotropinreleasing hormones in the pituitary of goldfish (Carassius auratus). Biol. Reprod. 40: 1152-1161. Hall, T.R. and Chadwick, A. 1984. Effects of synthetic mammalian thyrotropin-releasing hormone, somatostatin and dopamine on secretion of prolactin and growth hormone from amphibian and reptilian pituitary glands incubated in vitro. J. Endocrinol. 102: 175-180. Hall, T.R., Lam, S.R. and Harvey, S. 1985. Calcium control of growth hormone release from chicken pituitary glands in vitro. Gen. Comp. Endocrinol. 60: 70-74.
Hall, T.R., Harvey, S. and Scanes, C.G. 1986. Control of growth hormone secretion in the vertebrates: A comparative survey. Comp. Biochem. Physiol. 84A: 231-253. Hart, G.R., Ray, K.P. and Wallis, M. 1988. Mechanisms involved in the effects of TRH on GHRH-induced growth hormone release from ovine and bovine pituitary cells. Mol. Cell. Endocrinol. 56: 53-61. Harvey, S. 1990. Thyrotropin-releasing hormone: A growth hormone releasing factor. J. Endocrinol. 125: 345-358. Karber, G. 1931. Beitrug zur kollektiven behandlung pharmakologischen reihenversuche. Arch. Exp. Path. Pharmak. 162: 480-484. Mackenzie, O.S., Gould, D.R., Peter, R.E., Rivier, J. and Vale, W.W. 1984. Response of superfused goldfish pituitary fragments to mammalian and salmon gonadotropin-releasing hormone. Life Sci. 35: 2019-2026. Marchant, T.A., Fraser, R.A., Andrews, P.C. and Peter, R.E. 1987. The influences of mammalian and teleost somatostatin on the secretion of growth hormone from goldfish (Carassius auratusL.) pituitary fragments in vitro. Regul. Peptides 17: 41-52. Marchant, T.A., Chang, J.P., Nahorniak, C.S. and Peter, R.E. 1989. Evidence that gonadotropin-releasing hormone also functions as a growth hormone releasing factor in the goldfish. Endocrinology 124: 2509-2518. Peter, R.E., Nahorniak, C.S., Chang, J.P. and Crim, L.W. 1984. Gonadotropin release from the pars distalis of goldfish, Carassius auratus, transplanted beside the brain or into the brain ventricles: Additional evidence for gonadotropinrelease inhibitory factor. Gen. Comp. Endocrinol. 55: 337-346. Stojilkovic, C.A., Izumi, S. and Catt, K.J. 1988. Participation of voltage sensitive calcium channels in pituitary hormone release. J. Biol. Chem. 263: 13054-13061. Trudeau, V.L., Somoza, G.M., Nahorniak, C.S. and Peter, R.E. 1992. Interaction of estradiol with gonadotropinreleasing hormone and thyrotropin-releasing hormone in the control of growth hormone secretion in the goldfish. Neuroendocrinology (In press). Van Der Kraak, G., Suzuki, K., Peter, R.E., Itoh, H. and Kawauchi, H. 1991. Properties of common carp gonadotropin-I and gonadotropin-II. Gen. Comp. Endocrinol. 85: 217-229. Wigham, T. and Batten, T.F.C. 1984. In vitro effects of thyrotropin-releasing hormone and somatostatin on prolactin and growth hormone release by the pituitary of Poecilia latipinna. 1. An electrophoretic study. Gen. Comp. Endocrinol. 55: 444-449. Wong, A.O.-L., Chang, J.P. and Peter, R.E. 1992. Dopamine stimulates growth hormone release from the pituitary of goldfish, Carassiusauratus, through the dopamine D-1 receptors. Endocrinology 130: 1201-1210.
The regulatory effects of thyrotropin-releasing hormone on growth hormone secretion from the pituitary of common carp in vitro Xin-Wei Lin l , Hao-Ren Lin 1 and Richard E. Peter 2 'Department of Biology, Zhongshan University, Guangzhou, P.R. China. 510275; 2 Department of Zoology, University of Alberta, Edmonton, Alberta, Canada. T6G 2E9
Keywords: TRH, growth hormone, somatostatin, apomorphine, extracellular calcium, pituitary fragment, common carp
Abstract The effects of thyrotropin-releasing hormone (TRH) on growth hormone (GH) and gonadotropin (GtH) release, and the influences of somatostatin (SRIF), the dopamine agonist apomorphine (APO) and extracellular calcium on basal and TRH-induced GH release were examined using an in vitro perifusion system for pituitary fragments of common carp (Cyprinus carpio). Five minute pulses of different dosages of TRH stimulated a rapid and dose-dependent increase in GH release from the perifused pituitary fragments with an ED 50 of 9.7 + 2.3 nM. TRH was ineffective on GtH release. SRIF significantly inhibited basal and TRHinduced GH release from the perifused pituitary fragments, and the effects of SRIF were dose-dependent. APO induced a dose-dependent increase in basal and TRH-stimulated GH release from the perifused pituitary fragments. Increasing the concentrations of extracellular calcium from 0 mM to 1.25 mM resulted in an increase in basal and TRH-induced GH release. The high dose of calcium (6.25 mM) caused a slight decrease in basal and TRH-induced GH release compared with those at a concentration of 1.25 mM.
Resume Les effets de la thyrotropine (TRH) sur la scretion d'hormone de croissance (GH) et de gonadotropine (GTH), et de la somatostatine (SRIF), de l'apomorphine (APO), antagoniste dopaminergique, et du calcium extracellulaire sur les scr6tions basale et stimul6e de GH ont ete 6tudi6es in vitro par p6rifusion, de fragments d'hypophyses de carpe (Cyprinus carpio). Des applications de 5 minutes de TRH a diff6rentes concentrations induisent une stimulation rapide et dose d6pendante de la s6cr6tion de GH (ED 50 = 9.7 + 2.3 nM). Le TRH est sans effet sur la scr6tion de GTH. Le SRIF inhibe la scr6tion basale de GH ainsi que la rsponse hypophysaire a l'action du TRH. Son action est dose d6pendante. L'apomorphine induit une augmentation dose d6pendante de la s6cr6tion basale de GH et potentialise l'action du TRH sur la stimulation de la s6cr6tion de GH. Des effets equivalents sont induits par des concentrations croissantes de calcium extra cellulaire de 0 a 1.2 mM, alors qu'A une concentration de 6.25 mM des effets opposes sont obtenus. Introduction Thyrotropin-releasing hormone (TRH), a physiological stimulator of thyrotropin (TSH) and prolac-
tin secretion in mammals, has also been implicated as a stimulator of growth hormone (GH) release in certain pathological states and in perifusion with anterior pituitary cells of normal rat (for review, see
72 Harvey 1990). In birds, TRH is a major stimulatory factor of GH secretion (Hall et al. 1986; Harvey 1990), and TRH has also been reported to stimulate GH secretion in frogs (Hall and Chadwick 1984) and turtles (Denver and Licht 1989). A stimulatory effect of TRH on GH release has been suggested in the sailfin molly based on electrophoretic measurements of GH (Wigham and Batten 1984) and TRH stimulates GH release from perifused fragments of the goldfish pituitary (Trudeau et al. 1992). However, the interactions of somatostatin (SRIF), neurotransmitters or extracellular calcium on the actions of TRH on GH release in teleosts have not been investigated. In the present study, the effects of TRH on in vitro GH and gonadotropin (GtH) secretion in common carp were investigated. The influences of somatostatin (SRIF), the dopamine agonist apomorphine (APO) and extracellular calcium on basal and TRH-induced GH release were also examined. Materials and methods Common carp (Cyprinus carpio L.), 337-628 g of body weight, were purchased from local suppliers (Guangdong Province, P.R. China). Sexually recrudescing fish (male and female) were obtained in September (gonadosomatic index, GSI = 10.5 + 4.27o) for the dose-response experiments, and in November (GSI = 14.8 + 1.5%) for the other experiments. Fish were held in indoor recirculating 2501 aquaria at room temperature (18-28 0 C), with exposure to a natural photoperiod (Guangzhou, Guangdong Province, P.R. China). Experiments were conducted using a column perifusion system for pituitary fragments of common carp, similar to that used for goldfish pituitary fragments (Mackenzie et al. 1984; Habibi et al. 1989), with minor modifications. Briefly, the pituitaries were removed and fragments were prepared (< 1 mm3 ). Fragments equivalent to one half-pituitary were placed between two layers of Cytodex microcarrier beads (Sigma, St. Louis, MO, USA) in a 0.3 ml perifusion chamber. The fragments were perifused overnight (8-10h) with Medium 199 containing Hank's salts and Lglutamine (Gibco), 25 mM Hepes (Sigma) and 56
U/ml Nystatin (Sigma), at a flow rate of 5 ml/h. At 2h prior to the experiment, the medium was switched to Hank's balanced salt solution supplemented with 25 mM Hepes and 0.1% bovine serum albumin (Sigma) (HBSS, [Ca2 +] = 1.25 mM), or calcium-deficient HBSS in the fourth set of experiments at 19 + I°C, and the flow rate increased to 15 ml/h. Fractions were collected every 5 min., TRH (provided by the Laboratory of Reproductive Biology, Institute of Zoology, Academia Sinica) and SRIF (Sigma) were diluted with HBSS from stock solution (100 zM in HBSS), immediately before use. APO (Sigma) was dissolved in 0.8% NaCl with 0.1% sodium metabisulphite and diluted with HBSS just before use. In the dose-response study, the pituitary fragments were exposed to 5 min. pulses of increasing or decreasing concentrations of TRH (0.1, 1, 10, 100 and 1000 nM or 5, 50, 500, 5000 and 10000 nM) at 60 min. intervals. A total of 10 dosages of TRH were tested, each with six columns from three separate perifusion runs. The influences of APO on basal and TRHinduced GH release were studied in the second set of experiments. The pituitary fragments were exposed to three 5 min. pulses of increasing or decreasing concentrations of TRH (1, 10 and 100 nM) every 60 min. in combination with either HBSS alone, or 10, 100 or 1000 nM APO respectively. Each dose of TRH was tested with 4 column replicates from separate perifusion runs. The influences of SRIF on basal and TRHinduced GH release were studied in the third set of experiments. Three 5 min. pulses of 50 nM TRH were introduced at 60 min. intervals in the same perifusion column (four columns in each run, with two replicate runs). After the first pulse of TRH the pituitary fragments were perifused with 100 nM SRIF (in two columns) or in 10 nM SRIF (in another two columns), each lasting 60 min. and commencing 20 min. prior to the addition of the second TRH pulse. At the end of perifusion with SRIF, the fragments were perifused with HBSS alone for 25 min., and then the third pulse of TRH was introduced during continuous perifusion with HBSS. The influences of extracellular calcium on basal
73 and TRH-induced GH release were studied in the fourth set of experiments. The pituitary fragments were exposed to three 5 min. pulses of increasing or decreasing concentrations of TRH (1, 10 and 100 nM) every 60 min. in combination with Ca 2 +-deficient HBSS, or HBSS containing 0.25 mM, 1.25 mM and 6.25 mM Ca 2 + , respectively. Each dose of TRH was tested with 4 column replicates from separate perifusion runs. Calcium-deficient HBSS was HBSS without addition of Ca 2 + salts and containing 100 tM EGTA (Sigma). GtH and GH concentrations in the perifusates were determined by RIAs specific for common carp GtH-II (Peter et al. 1984; Van Der Kraak et al. 1992) and GH (Marchant et al. 1989), respectively. The hormone responses to each pulse of TRH were quantified, according to Habibi et al. (1989), and expressed as a percentage of the average basal hormone levels (prepulse) in the three fractions (15 min.) collected immediately preceding each pulse (% of prepulse). To calculate basal hormone levels, in the second set of experiments, the basal GH levels in the presence of different concentrations of APO were normalized as a percentage of the average GH levels over the 30 min. period before treatment with APO ( of pretreatment); in the third set of experiments, the basal GH levels in the presence of SRIF and HBSS alone preceding the second and third TRH pulses respectively were expressed as a percentage of the average GH level preceding the first pulse ( of 1st prepulse), and the first prepulses were quantified at 100%; in the fourth set of experiments, the average basal GH levels of prepulses in the presence of different concentrations of Ca 2 + were expressed as a percentage of the average GH levels over the 30 min. period during perifusion of calcium-deficient HBSS (o of pretreatment). The data were subjected to one-way analysis of variance followed by Duncan's multiple range test. The half-maximal effective dose (ED 50 ) value of TRH in stimulating GH release was calculated according to Karber (1931).
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TRH (nM) Fig. 1. Representative profile of GH release from perifused pituitary fragments of common carp in response to increasing doses of TRH administered as 5 min. pulses (black bar) every 60 min. (upper panel), and the dose-response curve of TRH in stimulation of GH and the effects of TRH on GtH secretion (lower panel). All values in the dose-response are mean + SEM of 6 observations from 3 separate perifusion runs. The ED 50 value of TRH on GH release is 9.7 + 2.3 nM. The average basal GtH and GH levels in these perifusion columns were 33.3 + 3.1 ng/ml and 126.2 + 34.7 ng/ml respectively.
Results Effects of TRH stimulating GH and GtH release Exposure of the sexually recrudescing common carp pituitary fragments to 5 min. pulses of increasing (upper panel, Fig. 1) or decreasing (not shown) doses of TRH resulted in a rapid and dosedependent stimulation of GH secretion. The ED 50 (mean approximate error) was estimated as 9.7 + 2.3 nM using 10 doses of TRH in order of increasing and decreasing concentration. TRH did not affect GtH release (lower panel, Fig. 1). Influences of APO on basal and TRH-induced GH secretion APO stimulated an increase in basal GH release (prepulse) in a dose-dependent manner (upper
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Fig. 2. Influences of APO on basal (upper panel) and TRHinduced (lower panel) GH release from perifused pituitary fragments of common carp. Each value is mean ± SEM of 4 observations from 4 individual perifusion runs. The average pretreatment GH level was 94.0 + 18.1 ng/ml. Similar basal GH levels (upper panel), or similar GH responses to the same dose of TRH in the presence of different doses of APO were identified by the same underscore (p < 0.05).
panel, Fig. 2). The high doses of APO (100, 1000 nM) significantly potentiated the actions of TRH (lower panel, Fig. 2); however, there were no significant differences between the GH release responses to pulses of TRH given alone and given in the presence of 10 nM APO. Influences of SRIF on basal and TRH-induced GH release Pulses of TRH (50 nM) alone stimulated a rapid GH release. The presence of 10 or 100 nM SRIF resulted in a significant reduction in basal and 50 nM TRH-induced GH release (upper panel, Fig. 3). SRIF (10 nM) partially blocked the TRH-induced GH release, and did not affect the GH release response to the third pulse of TRH (control) and the basal GH levels preceding this pulse (lower panel, Fig. 3); 100 nM SRIF completely blocked TRHinduced GH release (lower panel, Fig. 3) and significantly reduced the basal GH levels preceding the third TRH pulse (upper panel, Fig. 3).
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Fig. 3. Influences of SRIF on basal (upper panel) and TRHinduced (lower panel) GH release from perifused pituitary fragments of common carp. Each value represents mean + SEM of 4 observations from 2 separate perifusion runs. The average GH levels of the first prepulses was 108.6 + 26.1 ng/ml. The similar basal GH levels (upper panel), or GH release responses to TRH in the presence or absence of SRIF are identified by the same underscore (p < 0.05).
Influences of extracellular calcium on basal and TRH-induced GH secretion Basal GH levels were significantly increased with the elevation of concentrations of extracellular medium Ca 2 + from 0 mM to 1.25 mM; however, 6.25 mM Ca 2 + caused a slight decrease (insignificant) in basal levels compared to those at 1.25 mM 34.1 ng/ml) Ca 2 + . The basal GH levels (115.8 in the presence of 1.25 mM Ca 2 + (normal medium) were about four times the levels of basal GH (29.2 + 7.4 ng/ml) in the absence of Ca 2 + (Ca2+-deficient HBSS) (upper panel, Fig. 4). TRH-induced GH release responses were also significantly increased in the presence of increased concentrations of medium Ca 2 + (lower panel,
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TRH (nM) 2+ concentration on basal Fig. 4. Effects of extracellular Ca (upper panel) and TRH-induced (lower panel) GH release from perifused pituitary fragments of common carp. Each value is mean + SEM of 4 observations from 4 separate perifusion runs. 2 The average basal GH level in Ca +-deficient HBSS was 29.2 + 7.4 ng/ml. The similar basal GH levels (upper panel) or GH release responses to the same dose of TRH in the presence of different doses of Ca 2 + (lower panel) are identified by the same underscore (p < 0.05).
Fig. 4). In the absence of Ca 2 + , the GH-releasing actions of TRH were negligible. The high dose of Ca 2 + (6.25 mM) caused a reduction in the GH release responses to 10 nM, but not 1 and 100 nM TRH compared to the responses in the presence of 1.25 mM (lower panel, Fig. 4).
Discussion In the present study, we demonstrated that TRH can stimulate GH release via direct action on the pituitary of common carp. The potency of TRH in stimulating GH secretion from perifused pituitary fragments of common carp (sexually recrudescing, September) was similar to that in sexually regressed goldfish (Trudeau et al. 1992). TRH receptors in
brain and pituitary of goldfish have been partially characterized, with an apparent dissociation constant of 2-4 nM (Burt and Ajah 1984), which is in a similar range to the ED 50 estimate for TRHinduced GH secretion in the present study. The present results also demonstrate that TRH is ineffective on GtH release, indicating the GH response to TRH was specific. Present results further support the hypothesis that SRIF acts as the GH release-inhibiting factor in teleosts (Marchant et al. 1987). This is the first demonstration that the stimulatory actions of TRH on GH secretion in a teleost fish can be inhibited by SRIF, supporting the presence of a physiological role for TRH in GH secretion in teleosts. The influences of dopamine on GH-releasing actions of TRH in vertebrates are not clear. In the present study, the non-selective dopamine agonist APO induced a dose-dependent increase in basal GH release, similar to earlier findings in common carp (unpublished results) and goldfish (Chang et al. 1990). Dopamine acts via D-1 type receptors on somatotrophs to stimulate GH release in goldfish (Chang et al. 1990; Wong et al. 1992), and presumably is one of several GH-releasing factors. APO significantly increased TRH-stimulated GH release in the present study, suggesting the presence of dopaminergic regulation on GH secretion in common carp. In mammals and birds, several reports have suggested the involvement of a cAMP-independent, calcium-dependent mechanism underlying the stimulatory effects of TRH on GH secretion (Hart et al. 1988; Hall et al. 1985). Present results demonstrated that the mechanism of TRH action on in vitro GH release in common carp is Ca 2 + dependent, and the GH responses to TRH are somewhat reduced by a high concentration of Ca2 + (6.25 mM). Inhibition of neuropeptide-induced GH secretion by a supraphysiological dose of Ca 2 + was also reported for TRH in chicken (Hall et al. 1985) and for GHRH in mammals (Hart et al. 1988), but the mechanism for this inhibition remains to be determined. The present results also showed that basal GH release is dependent on extracellular Ca 2 +, consistent with the idea that extracellular Ca2 + plays an important role in mediating basal
76 GH secretion in the goldfish (Chang and De Leeuw 1990) and in other vertebrates (Hall et al. 1985; Stojilkovic et al. 1988).
Acknowledgements This work was supported by the Doctoral Fund from the National Education Committee of China to H.R. Lin and by grant 3-P-83-1101 from the International Development Research Center of Canada to H.R. Lin and R.E. Peter. We are grateful to J.P. Chang and H.R. Habibi for their help in the establishment of the pituitary perifusion system in H.R. Lin's laboratory. We also thank H. Kawauchi (Kitsato University, Japan) for the gift of purified common carp GH and GtH used in RIAs.
References cited Burt, P.R. and Ajah, M.A. 1984. TRH receptors in fish. Gen. Comp. Endocrinol. 53: 135-142. Chang, J.P. and De Leeuw, R. 1990. In vitro goldfish growth hormone responses to gonadotropin-releasing hormone: possible role of extracellular calcium and arachidonic acid metabolism? Gen. Comp. Endocrinol. 80: 155-164. Chang, J.P., Yu, K.L., Wong, A.O.L. and Peter, R.E. 1990. Differential actions of dopamine receptor subtypes on gonadotropin and growth hormone release in vitro in goldfish. Neuroendocrinology 51: 664-674. Denver, R.J. and Licht, P. 1989. Neuropeptides influences in vitro pituitary hormone secretion in hatchling turtles. J. Exp. Zool. 251: 306-315. Habibi, H.R., Marchant, T.A., Nahorniak, C.S., Van Der Loo, H., Peter, R.E., Rivier, J.E. and Vale, W.W. 1989. Functional relationship between receptor binding and biological activity for analogs of mammalian and salmon gonadotropinreleasing hormones in the pituitary of goldfish (Carassius auratus). Biol. Reprod. 40: 1152-1161. Hall, T.R. and Chadwick, A. 1984. Effects of synthetic mammalian thyrotropin-releasing hormone, somatostatin and dopamine on secretion of prolactin and growth hormone from amphibian and reptilian pituitary glands incubated in vitro. J. Endocrinol. 102: 175-180. Hall, T.R., Lam, S.R. and Harvey, S. 1985. Calcium control of growth hormone release from chicken pituitary glands in vitro. Gen. Comp. Endocrinol. 60: 70-74.
Hall, T.R., Harvey, S. and Scanes, C.G. 1986. Control of growth hormone secretion in the vertebrates: A comparative survey. Comp. Biochem. Physiol. 84A: 231-253. Hart, G.R., Ray, K.P. and Wallis, M. 1988. Mechanisms involved in the effects of TRH on GHRH-induced growth hormone release from ovine and bovine pituitary cells. Mol. Cell. Endocrinol. 56: 53-61. Harvey, S. 1990. Thyrotropin-releasing hormone: A growth hormone releasing factor. J. Endocrinol. 125: 345-358. Karber, G. 1931. Beitrug zur kollektiven behandlung pharmakologischen reihenversuche. Arch. Exp. Path. Pharmak. 162: 480-484. Mackenzie, O.S., Gould, D.R., Peter, R.E., Rivier, J. and Vale, W.W. 1984. Response of superfused goldfish pituitary fragments to mammalian and salmon gonadotropin-releasing hormone. Life Sci. 35: 2019-2026. Marchant, T.A., Fraser, R.A., Andrews, P.C. and Peter, R.E. 1987. The influences of mammalian and teleost somatostatin on the secretion of growth hormone from goldfish (Carassius auratusL.) pituitary fragments in vitro. Regul. Peptides 17: 41-52. Marchant, T.A., Chang, J.P., Nahorniak, C.S. and Peter, R.E. 1989. Evidence that gonadotropin-releasing hormone also functions as a growth hormone releasing factor in the goldfish. Endocrinology 124: 2509-2518. Peter, R.E., Nahorniak, C.S., Chang, J.P. and Crim, L.W. 1984. Gonadotropin release from the pars distalis of goldfish, Carassius auratus, transplanted beside the brain or into the brain ventricles: Additional evidence for gonadotropinrelease inhibitory factor. Gen. Comp. Endocrinol. 55: 337-346. Stojilkovic, C.A., Izumi, S. and Catt, K.J. 1988. Participation of voltage sensitive calcium channels in pituitary hormone release. J. Biol. Chem. 263: 13054-13061. Trudeau, V.L., Somoza, G.M., Nahorniak, C.S. and Peter, R.E. 1992. Interaction of estradiol with gonadotropinreleasing hormone and thyrotropin-releasing hormone in the control of growth hormone secretion in the goldfish. Neuroendocrinology (In press). Van Der Kraak, G., Suzuki, K., Peter, R.E., Itoh, H. and Kawauchi, H. 1991. Properties of common carp gonadotropin-I and gonadotropin-II. Gen. Comp. Endocrinol. 85: 217-229. Wigham, T. and Batten, T.F.C. 1984. In vitro effects of thyrotropin-releasing hormone and somatostatin on prolactin and growth hormone release by the pituitary of Poecilia latipinna. 1. An electrophoretic study. Gen. Comp. Endocrinol. 55: 444-449. Wong, A.O.-L., Chang, J.P. and Peter, R.E. 1992. Dopamine stimulates growth hormone release from the pituitary of goldfish, Carassiusauratus, through the dopamine D-1 receptors. Endocrinology 130: 1201-1210.
